In article <8qe1lk$e0d$1 at plutonium.btinternet.com>, "Patrick Druggan"
<Patrick.Druggan at btinternet.com> wrote:
> "Emir Khatipov" <ekhatipo at NOSPAMmidway.uchicago.edu> wrote in message
> news:Jmzx5.80$v3.1796 at uchinews...> > Hi,
> > I have problems with growing TOP10 transformants in M9 minimal medium.
> >
> > Could someone experienced in using M9 minimal medium advise me what is the
> > most appropriate way of preparing and handling/storing thiamine solution.
The TOP10 genotype doesn't list thi so thiamine in the medium would
be optional. Most recipes list it because "classic" strains (DH, JM
etc.) have mutations in thi. I don't think your problems is related
to the thiamine.
> > The TOP10 cells I am using cells that are L-lysine auxotrophs, so any
> > additional comments on lysine solution would be appreciated, too (making,
> > storing).
I use lysine solutions for yeast auxotrophs and they are stable for months
at 4degC.
> > I also know that I could add up to ~4% LB to facilitate growth, but I do
> not want to do that.
The other I've heard is to add casamino acids (some M9 recipes list
this ingredient at eg. 0.5%).
Are you familiar with bacteria growing in M9? They grow can *really*
slowly and colonies on plates are small.
I notice that TOP10 has a nupG mutation so maybe the cells are
nucleoside-deprived. You could try throwing some into the medium.
Bernard
--
Bernard P. Murray, PhD
bpmurray at cgl . ucsf . edu
Department of Cellular & Molecular Pharmacology, UCSF